Angiogenically effective unit dose of fgf and method of administering

ABSTRACT

The present invention provides a unit dose comprising 0.2 μg/kg to 36 μg/kg of a recombinant FGF or an angiogenically active fragment or mutein thereof. Also provided is a pharmaceutical composition comprising an angiogenically effective dose of an FGF or an angiogenically active fragment or mutein thereof, and a pharmaceutically acceptable carrier. Also provided is a method for treating a human patient for coronary artery disease, comprising administering into at least one coronary vessel of said patient a safe and angiogenically effective dose of a recombinant FGF of any of SEQ ID NOS:1-3, 5, 8-10, or 12-14, or an angiogenically active fragment or mutein thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/947,939, filed Nov. 17, 2010, which is a divisional of U.S.application Ser. No. 12/391,956, filed Feb. 24, 2009, now U.S. Pat. No.7,858,584, which is a divisional of U.S. application Ser. No.11/238,936, filed Sep. 29, 2005, now U.S. Pat. No. 7,511,019, which is acontinuation of U.S. application Ser. No. 10/131,965, filed Apr. 25,2002, now abandoned, which is a continuation of U.S. application Ser.No. 09/417,721, filed Oct. 13, 1999, now U.S. Pat. No. 6,451,303, whichclaims the benefit of U.S. Application Ser. No. 60/104,103, filed Oct.13, 1998, which applications are herein incorporated by reference intheir entirety.

FIELD OF THE INVENTION

The present invention is directed to a unit dose comprising an FGF or anangiogenically active fragment or mutein thereof for inducing cardiacangiogenesis in a human. The present invention is also directed to apharmaceutical composition comprising the unit dose of the FGF and to amethod for administering this pharmaceutical composition to a human toinduce cardiac angiogenesis while minimizing systemic risk to thepatient. The present invention is useful because the unit dose,pharmaceutical composition and the method for its administration providean alternative to surgical intervention for the treatment of coronaryartery disease (CAD) and may further provide an adjunct for reducingpost myocardial infarct (MI) injury in humans.

BACKGROUND OF THE INVENTION

The fibroblast growth factors (FGF) are a family of at least eighteenstructurally related polypeptides (named FGF-1 to FGF-18) that arecharacterized by a high degree of affinity for proteoglycans, such asheparin. The various FGF molecules range in size from 15-23 kD, andexhibit a broad range of biological activities in normal and malignantconditions including nerve cell adhesion and differentiation [Schubertet al., J. Cell Biol. 104:635-643 (1987)]; wound healing [U.S. Pat. No.5,439,818 (Fiddes)]; as mitogens toward many mesodermal and ectodermalcell types, as trophic factors, as differentiation inducing orinhibiting factors [Clements, et al., Oncogene 8:1311-1316 (1993)]; andas an angiogenic factor [Harada, J. Clin. Invest., 94:623-630 (1994)].Thus, the FGF family is a family of pluripotent growth factors thatstimulate to varying extents fibroblasts, smooth muscle cells,epithelial cells, and neuronal cells.

When FGF is released by normal tissues, such as in fetal development orwound healing, it is subject to temporal and spatial controls. However,many of the members of the FGF family are also oncogenes. Thus, in theabsence of temporal and spatial controls, they have the potential tostimulate tumor growth by providing angiogenesis.

Coronary artery disease (atherosclerosis) is a progressive disease inhumans wherein one or more coronary arteries gradually become occludedthrough the buildup of plaque. The coronary arteries of patients havingthis disease are often treated by balloon angioplasty or the insertionof stents to prop open the partially occluded arteries. Ultimately,these patients are required to undergo coronary artery bypass surgery atgreat expense and risk. It would be desirable to provide such patientswith a medicament that would enhance coronary blood flow so as topreclude the need to undergo bypass surgery.

An even more critical situation arises in humans when a patient suffersa myocardial infarction, wherein one or more coronary arteries orarterioles becomes completely occluded, such as by a clot. There is animmediate need to regain circulation to the portion of the myocardiumserved by the occluded artery or arteriole. If the lost coronarycirculation is restored within hours of the onset of the infarction,much of the damage to the myocardium that is downstream from theocclusion can be prevented. The clot-dissolving drugs, such as tissueplasminogen activator (tPA), streptokinase, and urokinase, have beenproven to be useful in this instance. However, as an adjunct to the clotdissolving drugs, it would also be desirable to also obtain collateralcirculation to the damaged or occluded myocardium by angiogenesis.

Accordingly, it is an object of the present invention to provide amedicament and a mode of administration that provides human patientswith cardiac angiogenesis during coronary artery disease and/or postacute myocardial infarction. More particularly, it is a further objectof the present invention to provide a therapeutic dose of an FGF and amode of administration to humans that provide the desired property ofcardiac angiogenesis, while minimizing adverse effects.

Many of the various FGF molecules have been isolated and administered tovarious animal models of myocardial ischemia with varying and oftentimes opposite results. According to Battler et al., “the canine modelof myocardial ischemia has been criticized because of the abundance ofnaturally occurring collateral circulation, as opposed to the porcinemodel, which ‘excels’ in its relative paucity of natural collateralcirculation and its resemblance to the human coronary circulation.”Battler et al., “Intracoronary Injection of Basic Fibroblast GrowthFactor Enhances Angiogenesis in Infarcted Swine Myocardium,” JACC,22(7): 2001-6 (December 1993) at page 2002, col.1. However, Battler etal., who administered bovine bFGF (i.e., FGF-2) to pigs in a myocardialinfarct model, considered the varying results that are obtained from oneanimal species to another, and expressly discloses that the divergentresults “thus emphasiz[e] the caution that must be exercised inextrapolating results from different animal models.” Battler et al., atpage 2005, col.1. Further, Battler points out that “the dosage and modeof administration of bFGF [i.e., bovine FGF-2] may have profoundimplications for the biologic effect achieved.” Battler, et al., at page2005, col.1. Thus, it is a further object of this invention to discovera dosage and a mode of administration of a fibroblast growth factor thatwould provide for the safe and efficacious treatment of CAD and/or postMI injury in a human patient. More generally, it is an object of thepresent invention to provide a pharmaceutical composition and method forinducing angiogenesis in a human heart.

SUMMARY OF THE INVENTION

The Applicants have discovered that a fibroblast growth factor, such asof SEQ ID NOS:1-3, 5, 8-9, or 12-14 or an angiogenically active fragmentor mutein thereof, when administered as a unit dose of about 0.2 μg/kgto about 36 μg/kg into one or more coronary vessels (IC) of a humanpatient in need of coronary angiogenesis, unexpectedly provides thehuman patient with a rapid and therapeutic cardiac angiogenesissufficient to obviate surgical intervention and results in anunexpectedly superior increase in the treated patient's exercisetolerance time (ETT). By way of comparison, angioplasty is considered atherapeutic success if it provides an increase in a patient's ETT ofgreater than 30 seconds compared to the placebo. By the term “cardiacangiogenesis” or “coronary angiogenesis,” as used herein, is meant theformation of new blood vessels, ranging in size from capillaries toarterioles which act as collaterals in coronary circulation.

FGFs that are suitable for use in the present invention include humanFGF-1 (SEQ ID NO:1), bovine FGF-1 (SEQ ID NO:2), human FGF-2 (SEQ IDNO:3), bovine FGF-2 (SEQ ID NO:5), human FGF-4 (SEQ ID NO:8), humanFGF-5 (SEQ ID NO:9), human FGF-6 (SEQ ID NO:10), human FGF-8 (SEQ IDNO:12), human FGF-9 (SEQ ID NO:13), and human FGF-98 (SEQ ID NO:14). Inone embodiment, FGF molecules are human FGF-1 (SEQ ID NO:1), bovineFGF-1 (SEQ ID NO:2), human FGF-2 (SEQ ID NO:3), bovine FGF-2 (SEQ IDNO:5), human FGF-4 (SEQ ID NO:8), and human FGF-5 (SEQ ID NO:9). In analternative embodiment, the FGF molecules are human FGF-6 (SEQ IDNO:10), murine FGF-8 (SEQ ID NO:12), human FGF-9 (SEQ ID NO:13) or humanFGF-98 (SEQ ID NO:14).

Typically, the angiogenically active fragments of the present inventionretain the distal two thirds of the mature FGF molecule (i.e., the twothirds of the molecule at the carboxy end that have the cell bindingsites). For convenience, the terms “human FGF,” “bovine FGF,” and murineFGF are used herein in abbreviated form as “hFGF,” “bFGF,” and “mFGF,”respectively.

The Applicants also discovered that a single unit dose of an FGF or anangiogenically active fragment thereof, when administered as a unit doseinto one or more coronary vessels (IC) of a human patient in need ofcoronary angiogenesis (e.g., a human patient with coronary arterydisease despite optional medical management), unexpectedly provides thehuman patient with a therapeutic benefit that is seen as early as twoweeks after the single unit dose is administered (as reflected insymptoms), and that lasts at least 60 days after the single unit dose isadministered (as reflected in ETT and the “Seattle Angina Questionnaire”(SAQ)). For example, when 28 human patients diagnosed as having CAD wereassessed by the SAQ both before and 57 days after being administered ICa single unit dose of 0.33 μg/kg to 48 μg/kg of FGF-2 of SEQ ID NO:5,the mean increase in their scores on the five criteria assessed rangedfrom 13 to 36 points, which is about 1.6-4.5 times greater than the 8point change which was considered to be “clinically significant” inalternative modes of treatment. See Table 2. When the scores of the 15first patients were broken down between those receiving a low dose (lessthan 2 μg/kg) and those receiving a higher dose (greater than or equalto 2 μg/kg) of FGF-2 of SEQ ID NO:5, and assessed by the SAQ, both doseswere found to provide scores that had “clinically significant” increasesranging from 12.3 to 58.1 and 10.9 to 32.1, respectively. Thus, whetherthe patients were administered the lower doses or the higher doses ofthe invention, their increased scores were about 1.4-7.2 times greaterthan the 8 point change which was considered to be “clinicallysignificant” in alternative modes of treatment. See Table 3.

As part of this study, MRI was performed on 23 human patients diagnosedwith CAD to assess ejection fraction, regional myocardial function andperfusion (delayed arrival zone). The patients were administered IC asingle unit dose of 0.33 μg/kg to 12 μg/kg of FGF-2 of SEQ ID NO:5.Their cardiac and coronary functions were objectively assessed bymagnetic resonance imaging (MRI) both before and after treatment. TheMRI results demonstrated significant improvement in regional wall motion(%) and wall thickening (%) during systole. The results also showed asignificant reduction in the delayed arrival zone (% LV). The resultsdid not demonstrate any significant change in ejection fraction (EF).Thus, the Applicants have demonstrated the clinical efficacy in humansof a single unit dose of an FGF when administered IC in accordance withthe present invention.

Accordingly, in one aspect, the Applicants' invention is directed to aunit dose of FGF comprising a safe and therapeutically effective amountof an FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or anangiogenically active fragment or mutein thereof. Typically, the safeand therapeutically effective amount comprises about 0.2 μg/kg to about36 μg/kg of an FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or anangiogenically active fragment or mutein thereof. In other embodiments,the safe and therapeutically effective amount of the unit dose comprises0.2 μg/kg to 2.0 μg/kg, 2.0 μg/kg to 20 μg/kg, or 20 μg/kg to 36 μg/kgof an FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or anangiogenically active fragment or mutein thereof. Expressed in absoluteterms for the majority of human CAD patients, the unit dose of thepresent invention comprises 0.008 mg to 6.1 mg, more typically 0.3 mg to3.5 mg, of the FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or anangiogenically active fragment or mutein thereof.

In another aspect, the present invention is directed to a pharmaceuticalcomposition comprising a safe and therapeutically effective amount of anFGF or an angiogenically active fragment or mutein thereof, and apharmaceutically acceptable carrier. Typically, the safe andtherapeutically effective amount of an FGF comprises about 0.2 μg/kg toabout 36 μg/kg of an FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14or an angiogenically active fragment or mutein thereof, and apharmaceutically acceptable carrier. In other embodiments of thepharmaceutical composition, the safe and therapeutically effectiveamount of an FGF comprises 0.2 μg/kg to 2 μg/kg, 2 μg/kg to 20 μg/kg, or20 μg/kg to 36 μg/kg of an FGF, such as an FGF of any one of SEQ IDNOS:1-3, 5, 8-10, or 12-14 or an angiogenically active fragment ormutein thereof, and a pharmaceutically acceptable carrier.

In yet another aspect, the present invention is directed to a method ofusing the above described unit dose or pharmaceutical composition totreat a human patient for CAD or to induce coronary angiogenesistherein. The method comprises administering into one or more coronaryvessels of a human patient in need of treatment for coronary arterydisease (or in need of angiogenesis) a safe and therapeuticallyeffective amount of a recombinant FGF or an angiogenically activefragment or mutein thereof. Typically, a portion of the safe andtherapeutically effective amount is administered to each of two coronaryvessels. More typically, the safe and therapeutically effective amountcomprises about 0.2 μg/kg to about 36 μg/kg of an FGF of any one of SEQID NOS:1-3, 5, 8-10, or 12-14 or an angiogenically active fragment ormutein thereof in a pharmaceutically acceptable carrier. In otherembodiments, the safe and therapeutically effective amount comprises 0.2μg/kg to 2 μg/kg, 2 μg/kg to 20 μg/kg, or 20 μg/kg to 36 μg/kg of theFGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or an angiogenicallyactive fragment or mutein thereof in a pharmaceutically acceptablecarrier.

Because FGF is a glycosoaminoglycan (e.g., heparin) binding protein andthe presence of a glycosoaminoglycan (also known as a “proteoglycan” ora “mucopolysaccharide”) optimizes activity and AUC, the IC dosages ofthe FGF of the present invention typically are administered within 20minutes of the IV administration of a glycosoaminoglycan, such as aheparin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a plot of the mean rFGF-2 plasma concentration versus time(hours) for six different doses of rFGF-2 (SEQ ID NO:5) administered byIC infusion in humans over a 20 minute period. The six doses of rFGF-2in FIG. 1 are 0.33 μg/kg, 0.65 μg/kg, 2 μg/kg, 6 μg/kg, 12 μg/kg, and 24μg/kg of lean body mass (LBM).

FIG. 2 is a plot of each individual patient's rFGF-2 area under thecurve (AUC) in pg·hr/ml for FIG. 1 for the six doses of rFGF-2, andshows the dose linearity of systemic rFGF-2 exposure following ICinfusion.

FIG. 3 is a plot of individual human patient rFGF-2 dose normalized AUCsas a function of the time of heparin administration in “minutes prior torFGF-2 infusion” and shows the influence of timing of heparinadministration on rFGF-2 AUC.

DETAILED DESCRIPTION OF THE INVENTION

The Applicants have discovered that single dose of an FGF or anangiogenically active fragment or mutein thereof, when administered in asafe and therapeutically effective amount into one or more coronaryvessels of a human patient diagnosed with CAD provides the patient witha safe and therapeutically efficacious treatment for the patient'scoronary artery disease that lasts at least 6 months before a furthertreatment is needed. In fact, the Applicants' method for treating CAD,when assessed by the standard objective criterion employed in the art(i.e., ETT), provided an unexpectedly superior increase of one and ahalf to two minutes in the treated patient's ETT. This comparesexceptionally well when compared to the increase of 30 seconds that isdeemed clinically significant for the current mode of treatment, i.e.,angioplasty.

By the phrase “safe and therapeutically effective amount” as used hereinin relation to FGF is meant an amount of an FGF or an angiogenicallyactive fragment or mutein thereof that when administered in accordancewith this invention, is free from major complications that cannot bemedically managed, and that provides for objective cardiac improvementin patients having symptoms of CAD despite optimum medical management.Thus, acute hypotension that can be managed by administration of fluids,with no other side effects is considered “safe” for the purpose of thisinvention. Typically, the safe and therapeutically effective amount ofan FGF comprises about 0.2 μg/kg to about 36 μg/kg of the FGF of any oneof SEQ ID NOS:1-3, 5, 8-10, or 12-14 or an angiogenically activefragment or mutein thereof.

Accordingly, the present invention has multiple aspects. In its firstaspect, the present invention is directed to a unit dose of the FGFmedicament that has produced unexpectedly superior results in treatingCAD in humans when compared to angioplasty. In particular, the unit dosecomprises a safe and therapeutically effective amount of an FGF or anangiogenically active fragment or mutein thereof. Typically, the unitdose comprises about 0.2 μg/kg to about 36 μg/kg of the FGF of any oneof SEQ ID NOS:1-3, 5, 8-10, or 12-14 or an angiogenically activefragment or mutein thereof. In other embodiments of the unit dose, thesafe and therapeutically effective amount comprises about 0.2 μg/kg toabout 2 μg/kg, about 2 μg/kg to about 20 μg/kg, or about 20 μg/kg toabout 36 μg/kg of the FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or12-14 or an angiogenically active fragment or mutein thereof. It isconvenient to provide the unit dose of the present invention in aformulation comprising in absolute terms from 0.008 mg to 6.1 mg of theFGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or an angiogenicallyactive fragment or mutein thereof. In this embodiment, the unit dosecontains a sufficient amount of FGF to accommodate dosing any one of themajority of CAD patients, ranging from the smallest patient (e.g., 40kg) at the lowest dosage (about 0.2 μg/kg) through the larger patients(e.g., 170 kg) at about the highest dosage (about 36 μg/kg). Moretypically, the unit dose comprises 0.3 mg to 3.5 mg of the FGF of anyone of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or an angiogenically activefragment or mutein thereof. The unit dose is typically provided insolution or lyophilized form containing the above referenced amount ofFGF and an effective amount of one or more pharmaceutically acceptablebuffers, stabilizers and/or other excipients as later described herein.

The active agent in the above described unit dose is a recombinant FGFor an angiogenically active fragment or mutein thereof. Typically, theactive agent of the unit dose is the FGF of any one of SEQ ID NOS:1-3,5, 8-10, or 12-14. More typically, the active agent in the unit dose ishFGF-1 (SEQ ID NO:1), bFGF-1 (SEQ ID NO:2), hFGF-2 (SEQ ID NO:3), bFGF-2(SEQ ID NO:5), hFGF-4 (SEQ ID NO:8) or hFGF-5 (SEQ ID NO:9). In analternative embodiment, the active agent in the unit dose is hFGF-6 (SEQID NO:10), mFGF-8 (SEQ ID NO:12), hFGF-9 (SEQ ID NO:13) or hFGF-98 (SEQID NO:14).

The amino acid sequences and methods for making many of the mammalianFGFs that are employed in the unit dose, pharmaceutical composition andmethod of the present invention are well known in the art. Inparticular, references disclosing the amino acid sequence andrecombinant expression of FGF 1-9 and FGF-98 are discussed sequentiallybelow.

FGF-1: The amino acid sequence of hFGF-1 (SEQ ID NO:1) and itsrecombinant expression are disclosed in U.S. Pat. No. 5,604,293(Fiddes), entitled “Recombinant Human Basic Fibroblast Growth Factor,”which issued on Feb. 18, 1997. See FIG. 2d of the '293 patent. Thisreference and all other references herein, whether cited before or afterthis sentence, are expressly incorporated herein by reference in theirentirety. The amino acid sequence and recombinant expression of bFGF-1(SEQ ID NO:2) are also disclosed in U.S. Pat. No. 5,604,293 (Fiddes)which has been incorporated herein by reference. See, FIG. 1 b of the'293 patent. Both hFGF-1 (SEQ ID NO:1) and bFGF-1 (SEQ ID NO:2) have 140amino acid residues. bFGF-1 differs from hFGF-1 at 19 residue positions:5(Pro→Leu), 21(His→Tyr), 31(Tyr→Val), 35(Arg→Lys), 40(Gln→Gly),45(Gln→Phe), 47(Ser→Cys), 51(Tyr→Ile), 54(Tyr→Val), 64(Tyr→Phe),80(Asn→Asp), 106(Asn→His), 109(Tyr→Val), 116(Ser→Arg), 117(Cys→Ser),119(Arg→Leu), 120(Gly→Glu), 125(Tyr→Phe), and 137(Tyr→Val). In mostinstances, the differences are conserved. Further, the differences atresidue positions 116 and 119 merely interchange the position of theArg.

FGF-2: The amino acid sequence of hFGF-2 (SEQ ID NO:3) and methods forits recombinant expression are disclosed in U.S. Pat. No. 5,439,818(Fiddes) entitled “DNA Encoding Human Recombinant Basic FibroblastGrowth Factor,” which issued on Aug. 8, 1995 (see FIG. 4 therein), andin U.S. Pat. No. 5,514,566 (Fiddes), entitled “Methods of ProducingRecombinant Fibroblast Growth Factors,” which issued on May 7, 1996 (seeFIG. 4 therein). The amino acid sequence of bFGF-2 (SEQ ID NO:5) andvarious methods for its recombinant expression are disclosed in U.S.Pat. No. 5,155,214, entitled “Basic Fibroblast Growth Factor,” whichissued on Oct. 13, 1992. When the 146 residue forms of hFGF-2 and bFGF-2are compared, their amino acid sequences are nearly identical with onlytwo residues that differ. In particular, in going from hFGF-2 to bFGF-2,the sole differences occur at residue positions 112(Thr→Ser) and128(Ser→Pro).

FGF-3: FGF-3 (SEQ ID NO:7) was first identified as an expression productof a mouse int-2 mammary tumor and its amino acid sequence is disclosedin Dickson et al., “Potential Oncogene Product Related to GrowthFactors,” Nature 326:833 (Apr. 30, 1987). FGF-3 (SEQ ID NO:7), which has243 residues when the N-terminal Met is excluded, is substantiallylonger than both FGF-1 (human and bovine) and FGF-2 (human and bovine).A comparison of amino acid residues for mFGF-3 (SEQ ID NO:7) relative tobFGF-1 (SEQ ID NO:2) and bFGF-2 (SEQ ID NO:5) is presented in overlapfashion in Dickson et al. (1987). When the amino acid sequence of mFGF-3(SEQ ID NO:7) is compared to bFGF-1 (SEQ ID NO 2): and bFGF-2 (SEQ IDNO:5), FGF-3 has 5 locations containing residue inserts relative to bothFGF-1 and FGF-2. The most significant of these inserts is a 12 and 14residue insert relative to FGF-2 and FGF-1, respectively, beginning atresidue position 135 of FGF-3. Allowing for the inserts, Dicksondiscloses that mFGF-3 has 53 residue identities relative to FGF-1 and 69residue identities relative to FGF-2. In addition, FGF-3 contains ahydrophobic N-terminal extension of 10 residues relative to theN-terminus of the signal sequence in both FGF-1 and FGF-2. Relative tothe C-terminus of bFGF-1 and bFGF-2, mFGF-3 contains an approximately 60residue extension. It is unlikely that the C-terminal extension ofmFGF-3 is necessary for activity. More likely, it is a moderator ofactivity by conferring receptor specificity on the FGF.

FGF-4: The amino acid sequence for the hst protein, now known as hFGF-4(SEQ ID NO:8), was first disclosed by Yoshida et al., “Genomic Sequenceof hst, a Transforming Gene Encoding a Protein Homologous to FibroblastGrowth Factors and the int-2-Encoded Protein,” PNAS USA, 84:7305-7309(October 1987). Including its leader sequence, hFGF-4 (SEQ ID NO:8) has206 amino acid residues. When the amino acid sequences of hFGF-4 (SEQ IDNO:7), hFGF-1 (SEQ ID NO:1), hFGF-2 (SEQ ID NO:3), and mFGF-3 (SEQ IDNO:7) are compared, residues 72-204 of hFGF-4 have 43% homology tohFGF-2 (SEQ ID NO:5); residues 79-204 have 38% homology to hFGF-1 (SEQID NO:1); and residues 72-174 have 40% homology to mFGF-3 (SEQ ID NO:7).A comparison of these four sequences in overlap form is shown in Yoshida(1987) at FIG. 3. Further, the Cys at residue positions 88 and 155 ofhFGF-4 are highly conserved among hFGF-1, hFGF-2, mFGF-3, and hFGF-4 andare found in a homologous region.

The two putative cell binding sites of hFGF-2 (SEQ ID NO:3) occur atresidue positions 36-39 and 77-81 thereof. See Yoshida (1987) at FIG. 3.The two putative heparin binding sites of hFGF-2 occur at residuepositions 18-22 and 107-111 thereof. See Yoshida (1987) at FIG. 3. Giventhe substantial similarity between the amino acid sequences for humanand bovine FGF-2, we would expect the cell binding sites for bFGF-2 (SEQID NO:5) to also be at residue positions 36-39 and 77-81 thereof, andthe heparin binding sites to be at residue positions 18-22 and 107-111thereof. In relation to hFGF-1 (SEQ ID NO:1), the putative cell bindingsites occur at residues 27-30 and 69-72, and the putative heparinbinding sites occur at residues 9-13 and 98-102. Insofar as bFGF-1 (SEQID NO:2) has the identical amino acids at residue positions 9-13, 27-30,69-72, and 98-102 as does hFGF-1 (SEQ ID NO:1), bFGF-1 would be expectedto have the same cell and heparin binding sites as does hFGF-1.

FGF-5: The amino acid sequence and method for cloning hFGF-5 (SEQ IDNO:15) are disclosed in Zhan, et al., “The Human FGF-5 Oncogene Encodesa Novel Protein Related to Fibroblast Growth Factors,” Molec. and Cell.Biol., 8(8):3487-3495 (August 1988). The Applicants also sequenced theFGF-5 and obtained the amino acid sequence of SEQ ID NO:9, whichdiffered from Zhan's sequence at residue position 236 (having a Lysinstead of the Zhan's Asn) and at residue position 243 (having a Proinstead of Zhan's Ser). Both hFGF-5 (SEQ ID NO:9) and hFGF-5 (SEQ IDNO:15) have 266 amino acid residues that include a leader sequence of 67residues upstream of the first residue of the FGF-2 of SEQ ID NO:5 and atail sequence that extends about 47 residues beyond the C-terminus ofhFGF-2. A comparison between the amino acid sequences of hFGF-1 (SEQ IDNO:1), hFGF-2 (SEQ ID NO:3), mFGF-3 (SEQ ID NO:7), hFGF-4 (SEQ ID NO:8),and FGF-5 (SEQ ID NO:9) is presented in FIG. 2 of Zhan (1988). In FIG. 2of Zhan, hFGF-1, hFGF-2, mFGF-3, and hFGF-4 are identified as aFGF(i.e., acidic FGF), bFGF (i.e., basic FGF), int-2, and hstKS3,respectively, i.e., by their original names. In the above referencedcomparison, two blocks of FGF-5 amino acid residues (90 to 180 and187-207) showed substantial homology to FGF 1-4, i.e., 50.4% with FGF-4,47.5% with FGF-3, 43.4% with FGF-2 and 40.2% with hFGF-1. See Zhan(1988) at FIG. 2. U.S. Pat. No. 5,155,217 (Goldfarb) and U.S. Pat. No.5,238,916 (Goldfarb), which correspond to the Zhan publication, refer tothe FGF-5 of Zhan as FGF-3. However, the art (as evidenced by Coulierbelow) has come to recognize that the hFGF of Zhan (and Goldfarb) asFGF-5 and not as FGF-3. The two Goldfarb patents contain the same aminoacid sequence for hFGF-5 (SEQ ID NO:15) as reported above by Zhan.

FGF-6: The amino acid sequence and method for cloning hFGF-6 (SEQ IDNO:10) are disclosed in Coulier et al., “Putative Structure of the FGF-6Gene Product and Role of the Signal Peptide,” Oncogene 6:1437-1444(1991). hFGF-6 is one of the largest of the FGFs, having 208 amino acidresidues. When the amino acid sequences of human FGF-1, FGF-2, FGF-3,FGF-4, FGF-5, FGF-6, and FGF-7 are compared, there are strongsimilarities in the C-terminal two-thirds of the molecules(corresponding e.g., to residues 78-208 of hFGF-6 (SEQ ID NO:10)). Inparticular, 23 residues, including two cysteines (at positions 90-157 ofhFGF-6 of SEQ ID NO:10) were identical between the seven members of thefamily. This number increases to 33 residues when conserved amino acidresidues are considered. The overall similarities between these sevenhuman FGFs range from 32 to 70% identical residues and 48 to 79%conserved residues for the C-terminal two-thirds of the molecules. Thesequence comparisons, relative to FGF-6, are shown in Table 1 herein.

TABLE 1 Amino Acid Sequence Comparison of hFGF-6 With Other hFGFsIdentical Conserved Identical Conserved SEQ ID NO: Residues* Residues**Residues* (%) Residues** (%) hFGF-4 8 91 103 70 79 hFGF-5 9 64 82 49 63hFGF-3 7 55 78 42 60 hFGF-2 3 54 69 42 53 hFGF-7 11 47 68 36 52 hFGF-1 142 62 32 48 *Number and percentages of identical or conserved residueswere calculated for the C-terminal two-thirds of the hFGF6 molecule(residues 78-208). **Conserved residues are defined according to thestructure-genetic matrix of Feng et al., J. Mol. Evol., 21: 112-125(1985).

Referring to Table 1, FGF-6 has the highest correspondence (91 identicalresidues/103 conserved residues) with FGF-4. This amounts to 70%identical and 79% conserved residues. hFGF-6 (SEQ ID NO:10) differedmost from hFGF-3 (SEQ ID NO:2); hFGF-2 (SEQ ID NO:3), hFGF-7 (SEQ IDNO:11), and FGF-1 (SEQ ID NO:1), with 42, 42, 36, and 32 identicalresidues, respectively.

An overlayed comparison of the amino acid sequences of FGFs 1-7 is shownin FIG. 3 of incorporated Coulier (1991). FIG. 3 of Coulier shows thatwhen the C-terminal two thirds of the FGF molecules are aligned, thereare 23 residue positions wherein the residues from all seven FGF membersare identical. There are also ten residue positions wherein residuesfrom all seven FGF members are conserved. Coulier (1991) at FIG. 3. Incombination, these identical and conserved residues form about 6locations of three to five residues on the terminal two thirds of eachof the FGFs 1-7, wherein three to five residues are grouped together inall seven species of human FGF (i.e., hFGF 1-7).

FGF-7: The amino acid sequence of hFGF-7 (SEQ ID NO:11) is disclosed inMiyamoto, et al., “Molecular Cloning of a Novel Cytokine cDNA Encodingthe Ninth Member of the Fibroblast Growth Factor Family, Which Has aUnique Secretion Property,” Mol. and Cell. Biol. 13(7):4251-4259 (1993).In Miyamoto, the hFGF-7 was referred to by its older name KGF. Asreflected in SEQ ID NO:11, FGF-7 has 191 amino acid residues. Miyamotocompared hFGF-7 (SEQ ID NO:11) to hFGF 1-6 and hFGF-9 shows that thecarboxy terminal two thirds of the FGF-7 has comparable homology withthe distal two thirds of the other members of the group. See Miyamoto(1993) at page 4254 (FIG. 2).

FGF-8: The amino acid sequence of mFGF-8 (SEQ ID NO:12) and a method forits recombinant expression are disclosed in Tanaka et al., “Cloning andCharacterization of an Androgen-induced Growth Factor Essential for theGrowth of Mouse Mammary Carcinoma Cells,” PNAS USA, 89:8928-8932 (1992).The mFGF-8 of Tanaka has 215 amino acid residues. MacArthur, et al.,“FGF-8 Isoforms Activate Receptor Splice Forms that Are Expressed inMesenchymal Regions of Mouse Development,” Development, 121:3603-3613(1995) discloses that FGF-8 has 8 different isoforms that differ at themature N-terminus but that are identical over the C-terminal region. The8 isoforms arise because FGF-8 has 6 exons of which the first four(which correspond to the first exon of most other FGF genes) result inalternative splicing.

FGF-9: The amino acid sequence of hFGF-9 and a method for itsrecombinant expression are disclosed in Santos-Ocampo, et al.,“Expression and Biological Activity of Mouse Fibroblast Growth Factor,”J. Biol. Chem., 271(3):1726-1731 (1996). Notwithstanding its title,Ocampo discloses the amino acid sequence of both hFGF-9 (SEQ ID NO:13)and mFGF-9. Both the human and murine FGF-9 molecules have 208 aminoacid residues and sequences that differ by only two residues. Inparticular, the hFGF-9 has Asn and Ser at residues 9 and 34,respectively, whereas the mFGF-9 has Ser and Asn, respectively. FGF-9has complete preservation of the conserved amino acids that define theFGF family. Santos-Ocampo (1996) at page 1726. Half-maximal activationof FGF-9 is seen at 185 ng/ml heparin, whereas half-maximal activationof FGF-1 is seen at 670 ng/ml heparin. Santos-Ocampo (1996) at page1730. When compared to FGF-1, both FGF-2 and FGF-9 require lower heparinconcentrations for optimal activity.

FGF-98: The amino acid sequence of hFGF-98 (SEQ ID NO:14) and a methodfor its recombinant expression are disclosed in provisional patentapplication Ser. No. 60/083,553 which is hereby incorporated herein byreference in its entirety. hFGF-98, which is also known as hFGF-18, has207 amino acid residues. Thus, hFGF-6 (207 residues), hFGF-9 (208residues), and hFGF-98 (207 residues) are similar in size.

FGFs differentially bind to and activate one or more of four relatedtransmembrane receptors which in turn mediate a biological response. TheFGF receptors (“FGFR”) are members of the tyrosine kinase receptorsuperfamily. The extracellular domain of the FGFR comprises 2-3immunoglobulin-like (“IG-like”) domains that are differentiallyexpressed as a result of alternative splicing. Another alternativesplicing event can also alter the sequence of the carboxy-terminal halfof the Ig-like domain III without altering the reading frame.Santos-Ocampo (1996). The two splice forms, which are referred to as “b”and “c”, occur for FGFRs 1, 2, 3 but not 4. A more detailed descriptionof the FGFR is found in Mathieu, et al, “Receptor Binding and MitogenicProperties of Mouse Fibroblast Growth Factor 3,” J. Biol. Chem.,270(41):24197-24203 (1995). The ability of FGF 1-9 to differentiallystimulate FGFRs was receptor dependent as reported by Ornitz et al., J.Biol. Chem., 271(25):15292-15297 (1996). In Ornitz, the cell line BaF3was divided into fractions and each fraction was transfected to expressone of the following FGF receptors: FGFR1b, FGFR1c, FGFR2b, FGFR2c,FGFR3b, FGFR3c, and FGF4 (minus one Ig-like domain). Thereafter, thetransformed cell lines were exposed to one of FGF 1-9 (5 nM) and heparin(2 μg/ml) as a cofactor. The mitogenic response was then measured byincorporation of [³H] thymidine. The results in cpm are as follows:

1. FGFR1b: similar mitogenic responses were produced by hFGF-1 (32,000cpm) and hFGF-2 (28,000 cpm) with the next highest responses by mFGF-3(about 16,000 cpm) and hFGF-4 (15,000 cpm);

2. FGFR1c: similar mitogenic responses were produced by hFGF-1, hFGF-2,hFGF-4, hFGF-5, and hFGF-6 (about 36,000 cpm), with mFGF-9 producing theonly other significant response (about 19,000 cpm);

3. FGFR2b: best mitogenic responses were by hFGF-7 (14,000 cpm), hFGF-1(12,500 cpm), and mFGF-3 (9,500 cpm);

4. FGFR2c: best mitogenic responses were by hFGF-4 (21,000 cpm), mFGF-9(20,000 cpm), hFGF-6 (16,500 cpm), hFGF-1 (16,000 cpm), hFGF-2 (14,500cpm), hFGF-5 (9,500 cpm), and mFGF-8 (9,000 cpm);

5. FGFR3b: mitogenic responses only by hFGF-1 (37,000 cpm) and mFGF-9(26,000 cpm);

6. FGFR3c: best mitogenic responses by hFGF-1 (39,000 cpm), hFGF-2(34,000 cpm), hFGF-4 (33,000 cpm), mFGF-8 (32,500 cpm), mFGF-9 (31,000cpm), hFGF-5 (16,000 cpm), and hFGF-6 (13,000 cpm);

7. FGFR4Δ: best mitogenic responses by hFGF-2 (29,000 cpm), hFGF-4 andhFGF-6 (27,000 cpm), mFGF-8 (25,000 cpm), mFGF-1 (24,000 cpm), andhFGF-9 (20,000 cpm) with all others being 6,000 cpm or less.

As reflected above, only FGF-1 induces a significant mitogenic responsein all of the receptors tested. Thus, FGF-1 can be thought of as auniversal ligand with N- and C-terminal additions to the molecule givingrise to receptor specificity associated with the other FGF. Given thepotential for diverse responses in vivo by systemically administeredFGF, the present invention minimizes the potential for systemicresponses by localized administration, and by discovering theappropriate dosage for the localized administration, i.e., byadministering a therapeutically effective amount of an FGF into at leastone coronary artery of a patient in need of treatment for CAD.

In the Examples that follow, bFGF-2 (SEQ ID NO:5) was administered invivo to rats, pigs, and humans, and tested for angiogenic activity. ThebFGF-2 of the Examples was made as described in U.S. Pat. No. 5,155,214.In the '214 patent, a DNA of SEQ ID NO:4, which encodes a bFGF(hereinafter “FGF-2”) of SEQ ID NO:5, is inserted into a cloning vector,such as pBR322, pMB9, Col E1, pCRI, RP4 or λ-phage, and the cloningvector is used to transform either a eukaryotic or prokaryotic cell,wherein the transformed cell expresses the FGF-2. In one embodiment, thehost cell is a yeast cell, such as Saccharomyces cerevisiae. Theresulting full-length FGF-2 that is expressed has 146 amino acids inaccordance with SEQ ID NO:5. Although the FGF-2 of SEQ ID NO:5 has fourcysteines, i.e., at residue positions 25, 69, 87, and 92, there are nointernal disulfide linkages. ['214 at col. 6, lines 59-61.] However, inthe event that cross-linking occurred under oxidative conditions, itwould likely occur between the residues at positions 25 and 69.

The FGF-2 of SEQ ID NO:5, which is of bovine origin, like thecorresponding human FGF-2 is initially synthesized in vivo as apolypeptide having 155 amino acid residues. Abraham et al. “Human BasicFibroblast Growth Factor: Nucleotide Sequence and Genomic Organization,”EMBO J., 5(10):2523-2528 (1986). When compared to the full-length155-residue bovine FGF-2 of Abraham, Applicants' FGF-2 of SEQ ID NO:5lacks the first nine amino acid residues, Met Ala Ala Gly Ser Ile ThrThr Leu (SEQ ID NO:3), at the N-terminus of the correspondingfull-length molecule. As discussed above, the FGF-2 of SEQ ID NO:5differs from human FGF-2 in two residue positions. In particular, theamino acids at residue positions 112 and 128 of the bFGF-2 of SEQ IDNO:5 are Ser and Pro, respectively, whereas in hFGF-2, they are Thr andSer, respectively. Given this substantial structural identity, the invivo clinical results provided in the Examples and discussed elsewhereherein on bFGF-2 (SEQ ID NO:5) should be directly applicable to hFGF-2(SEQ ID NO:3).

The recombinant bFGF-2 (SEQ ID NO:5) of the Examples was purified topharmaceutical quality (98% or greater purity) using the techniquesdescribed in detail in U.S. Pat. No. 4,956,455, entitled “BovineFibroblast Growth Factor” which issued on Sep. 11, 1990 and which wasincorporated herein by reference in its entirety. In particular, thefirst two steps employed in the purification of the recombinant bFGF-2of Applicants' unit dose are “conventional ion-exchange and reversephase HPLC purification steps as described previously.” [U.S. Pat. No.4,956,455, citing to Bolen et al., PNAS USA 81:5364-5368 (1984).] Thethird step, which the '455 patent refers to as the “key purificationstep” ['455 at col. 7, lines 5-6], is heparin-SEPHAROSE® affinitychromatography, wherein the strong heparin binding affinity of the FGF-2is utilized to achieve several thousand-fold purification when elutingat approximately 1.4 M and 1.95 M NaCl ['455 at col. 9, lines 20-25].Polypeptide homogeneity was confirmed by reverse-phase high pressureliquid chromatography (RP-HPLC). Buffer exchange was achieved bySEPHADEX® G-25(M) gel filtration chromatography.

In addition to the FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14,the active agent in the unit dose of the present invention alsocomprises an “angiogenically active fragment thereof.” By the term“angiogenically active fragment thereof” is meant a fragment of any oneof the FGF of SEQ ID NOS:1-3, 5, 8-10, or 12-14 that has about 80% ofthe residues sequence of SEQ ID NO:5 and that retains the angiogeniceffect of the corresponding mature FGF. A common truncation is theremoval of the N-terminal methionine, using well known techniques suchas treatment with a methionine aminopeptidase. A second desirabletruncation comprises the FGF without its leader sequence. Those skilledin the art recognize the leader sequence as the series of hydrophobicresidues at the N-terminus of a protein that facilitate its passagethrough a cell membrane but that are not necessary for activity and thatare not found on the mature protein.

Preferred truncations are determined relative to hFGF-2 (or theanalogous bFGF-2). As a general rule, the amino acid sequence of an FGFis aligned with FGF-2 to obtain maximum homology. Portions of the FGFthat extend beyond the corresponding N-terminus of the aligned FGF-2(SEQ ID NO:3) are suitable for deletion without adverse effect.Likewise, portions of the FGF that extend beyond the C-terminus of thealigned FGF-2 (SEQ ID NO:3) are also capable of being deleted withoutadverse effect.

Fragments of FGF that are smaller than those described above are alsowithin the scope of the present invention so long as they retain thecell binding portions of FGF and at least one heparin binding segment.As already discussed above, the heparin binding segments of FGF-2 (humanor bovine) occur at residues 18-22 and 107-111, whereas the cell bindingportions occur at residues 36-39 and 77-81. For example, it is wellknown in the art that N-terminal truncations of bFGF-2 do not eliminateits angiogenic activity in cows. In particular, the art disclosesseveral naturally occurring and biologically active fragments of bFGF-2of SEQ ID NO:5 that have N-terminal truncations relative to the bFGF-2of SEQ ID NO:5. An active and truncated bFGF-2 having residues 12-146 ofSEQ ID NO:5 was found in bovine liver and another active and truncatedbFGF-2, having residues 16-146 of SEQ ID NO:5 was found in the bovinekidney, adrenal glands, and testes. [See U.S. Pat. No. 5,155,214 at col.6, lines 41-46, citing to Ueno, et al., Biochem and Biophys Res. Comm.,138:580-588 (1986).] Likewise, other fragments of the bFGF-2 of SEQ IDNO:5 that are known to have FGF activity are FGF-2 (24-120)-OH and FGF-2(30-110)-NH₂. [U.S. Pat. No. 5,155,214 at col. 6, lines 48-52.] Theselatter fragments retain both of the cell binding portions of bFGF-2 (SEQID NO:5) and one of the heparin binding segments (residues 107-111).Accordingly, the angiogenically active fragments of an FGF typicallyencompass those terminally truncated fragments of an FGF that whenaligned to an FGF-2 to maximize homology, have at least residues thatcorrespond to residues 30-110 of bFGF-2 of SEQ ID NO:5 (or the hFGF-2 ofSEQ ID NO:3); more typically, at least residues that correspond toresidues 18-146 of bFGF-2 of SEQ ID NO:5.

The unit dose of the present invention also comprises an “angiogenicallyactive . . . mutein” of the FGF of any one of SEQ ID NOS:1-3, 5, 8-10,or 12-14. By the term “angiogenically active . . . mutein” is meant amutated form of the FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14that structurally retains at least 80%, preferably 90%, of the residuesof any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 in their respectivepositions, and that functionally retains the angiogenic activity of theFGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14. Preferably, themutations are “conservative substitutions” using L-amino acids, whereinone amino acid is replaced by another biologically similar amino acid.Examples of conservative substitutions include the substitution of onehydrophobic residue such as Ile, Val, Leu, Pro, or Gly for another, orPhe←→Tyr, Ser←→Thr, or the substitution of one polar residue foranother, such as between Arg and Lys, between Glu and Asp, or betweenGln and Asn, and the like. Generally, the charged amino acids areconsidered interchangeable with one another. However, to make thesubstitution more conservative, one takes into account both the size andthe likeness of the charge, if any, on the side chain. Other suitablesubstitutions include the substitution of serine for one or both of thecysteines at residue positions which are not involved in disulfideformation, such as residues 87 and 92 in hFGF-2 (SEQ ID NO:3) or bFGF-2(SEQ ID NO:5). Preferably, substitutions are introduced at theN-terminus, which is not associated with angiogenic activity. However,as discussed above, conservative substitutions are suitable forintroduction throughout the molecule.

One skilled in the art, using art known techniques, is able to make oneor more point mutations in the DNA encoding an FGF of any one of SEQ IDNOS:1-3, 5, 8-10, or 12-14 to obtain expression of an FGF polypeptidemutein (or fragment mutein) having angiogenic activity for use withinthe unit dose, compositions and method of the present invention. Toprepare an angiogenically active mutein of the FGF of any one of SEQ IDNOS:1-3, 5, 8-10, or 12-14, one uses standard techniques forsite-directed mutagenesis, as known in the art and/or as taught inGilman, et al., Gene, 8:81 (1979) or Roberts, et al., Nature, 328:731(1987), to introduce one or more point mutations into the cDNA thatencodes the FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14.

In a second aspect, the present invention is directed to apharmaceutical composition comprising a safe and an angiogenicallyeffective dose of an FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14or an angiogenically active fragment or mutein thereof, and apharmaceutically acceptable carrier. Typically, the safe andangiogenically effective dose of the pharmaceutical composition of thepresent invention is in a form and a size suitable for administration toa human patient and comprises (i) 0.2 μg/kg to 36 μg/kg of an FGF of anyone of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or an angiogenically activefragment or mutein thereof, (ii) and a pharmaceutically acceptablecarrier. In other embodiments, the safe and angiogenically effectivedose comprises 0.2 μg/kg to 2 μg/kg, 2 μg/kg to 20 μg/kg, or 20 μg/kg to36 μg/kg of the FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 oran angiogenically active fragment or mutein thereof, and apharmaceutically acceptable carrier.

By the term “pharmaceutically acceptable carrier” as used herein ismeant any of the carriers or diluents known in the art for thestabilization and/or administration of a proteinaceous medicament, suchas the FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 disclosedherein, that does not itself induce the production of antibodies harmfulto the individual receiving the composition, and which may beadministered without undue toxicity. Within another aspect of theinvention, pharmaceutical compositions are provided, comprising arecombinant FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or anangiogenically active fragment or mutein thereof in combination with apharmaceutically acceptable carrier or diluent. Such pharmaceuticalcompositions may be prepared either as a liquid solution, or as a solidform (e.g., lyophilized) which is dissolved in a solution prior toadministration. In addition, the composition may be prepared withsuitable carriers or diluents for IC injection or administration.Pharmaceutically acceptable carriers or diluents are nontoxic to a humanrecipient at the dosages and concentrations employed. Representativeexamples of suitable carriers or diluents for injectable or infusiblesolutions include sterile water or isotonic saline solutions, which arepreferably buffered at a suitable pH (such as phosphate-buffered salineor Tris-buffered saline), and optionally contain mannitol, dextrose,glycerol, ethanol, and/or one or more polypeptides or proteins such ashuman serum albumin (HSA). Stabilizers, such as trehalose, thioglycerol,and dithiothreitol (DTT), may also be added.

A typical pharmaceutical composition comprises 0.001 to 10 mg/ml, moretypically 0.03 to 0.5 mg/ml, of a rFGF of any one of SEQ ID NOS:1-3, 5,8-10, or 12-14 or an angiogenically active fragment or mutein thereof,10 mM thioglycerol, 135 mM NaCl, 10 mM Na citrate, and 1 mM EDTA, pH 5.A suitable diluent or flushing agent for the above described compositionis any of the above described carriers. Typically, the diluent is thecarrier solution itself comprising 10 mM thioglycerol, 135 mM NaCl, 10mM Na citrate, and 1 mM EDTA, pH 5. The rFGF of any one of SEQ IDNOS:1-3, 5, 8-10, or 12-14 or an angiogenically active fragment ormutein thereof is unstable for long periods of time in liquid form. Tomaximize stability and shelf life, the pharmaceutical composition of thepresent invention comprising an effective amount of rFGF of any one ofSEQ ID NOS:1-3, 5, 8-10, or 12-14 or an angiogenically fragment ormutein thereof, in a pharmaceutically acceptable aqueous carrier shouldbe stored frozen at −60° C. When thawed, the solution is stable for 6months at refrigerated conditions. A typical unit dose would compriseabout 5-10 ml of the above described composition having 1.5-8 mg of theFGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14.

In another embodiment, the pharmaceutical composition comprises a unitdose of FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or anangiogenically active fragment or mutein thereof in lyophilized(freeze-dried) form. In this form, the unit dose of FGF would be capableof being stored at refrigerated temperatures for substantially longerthan 6 months without loss of therapeutic effectiveness. Lyophilizationis accomplished by the rapid freeze drying under reduced pressure of aplurality of vials, each containing a unit dose of the FGF of thepresent invention therein. Lyophilizers, which perform the abovedescribed lyophilization, are commercially available and readilyoperable by those skilled in the art. Prior to administration to apatient, the lyophilized product is reconstituted to a knownconcentration, preferably in its own vial, with an appropriate sterileaqueous diluent, typically 0.9% (or less) sterile saline solution, or acompatible sterile buffer, or even sterile deionized water. Dependingupon the weight of the patient in kg, a single dose comprising from 0.2μg/kg to 36 μg/kg of the FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or12-14 or an angiogenically active fragment or mutein thereof iswithdrawn from the vial as reconstituted product for administration tothe patient. Thus, an average 70 kg man that is being dosed at 24 μg/kg,would have a sufficient volume of the reconstituted product withdrawnfrom the vial to receive an IC infusion of (70 kg×24 μg/kg) 1680 μg(i.e., 1.680 mg).

In its third aspect, the present invention is directed to a method fortreating a patient in need of treatment for CAD or MI, using the abovedescribed unit dose or pharmaceutical composition to treat a humanpatient for coronary artery disease (CAD). In particular, the presentinvention is directed to a method for treating a human patient forcoronary artery disease, comprising administering a safe andtherapeutically effective amount of a recombinant FGF of any one of SEQID NOS:1-3, 5, 8-10, or 12-14 or an angiogenically active fragment ormutein thereof to one or more, typically two, coronary vessels of ahuman patient in need of treatment for coronary artery disease. Apreferred coronary vessel is the coronary artery, although graftedsaphenous veins and grafted internal mammary arteries, as provided bycoronary angioplasty, are also suitable.

The method of the present invention provides clinical treatment of theunderlying condition (i.e., CAD or MI) and not merely a treatment of thesymptoms, such as provided by nitrates. Typically, the safe andtherapeutically effective amount of the method of the present inventioncomprises 0.2 μg/kg to 36 μg/kg of the FGF of any one of SEQ ID NOS:1-3,5, 8-10, or 12-14 or an angiogenically active fragment or mutein thereofin a pharmaceutically acceptable carrier. In other embodiments, the safeand therapeutically effective amount comprises 0.2 μg/kg to 2 μg/kg, 2μg/kg to 20 μg/kg, or 20 μg/kg to 36 μg/kg of the FGF of any one of SEQID NOS:1-3, 5, 8-10, or 12-14 or an angiogenically fragment or muteinthereof in a pharmaceutically acceptable carrier. In absolute terms, thesafe and therapeutically effective amount is about 0.008 mg to about 6.1mg of the FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or anangiogenically fragment or mutein thereof; more typically, 0.3 mg to 3.5mg of the FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or anangiogenically fragment or mutein thereof.

The therapeutically effective amount of the rFGF-2 of the presentinvention is administered to at least one coronary vessel of a humanpatient diagnosed with CAD, symptomatic despite optimal medicalmanagement, using standard cardiac catheterization techniques alreadyknown and used in the art for the intracoronary administration ofmedicaments, e.g., thrombolytics, streptokinase, or radio-opaque dyes ormagnetic particles used to visualize the coronary arteries. By way ofexample, a coronary catheter is inserted into an artery (e.g., femoralor subclavian) of the patient in need of treatment and the catheter ispushed forward, with visualization, until it is positioned in theappropriate coronary vessel of the patient to be treated. Using standardprecautions for maintaining a clear line, the pharmaceutical compositionin solution form is administered by infusing the unit dose substantiallycontinuously over a period of 10 to 30 minutes. Although thepharmaceutical composition of the invention could be administered over alonger period of time, the Applicants perceive no benefit and apotentially increased risk of thrombosis in doing so. Typically, aportion (e.g., one half) of the unit dose is administered in a firstcoronary vessel. Then, the catheter is repositioned into a secondsecondary coronary vessel and the remainder of the unit dose isadministered with flushing of the catheter. Using the above-describedrepositioning procedure, portions of the unit dose may be administeredto a plurality of coronary vessels until the entire unit dose has beenadministered. After administration, the catheter is withdrawn usingconventional art known protocols. Signs of coronary angiogenesis areapparent in a matter of days following IC administration of the unitdose. Therapeutic benefit is seen as early as two weeks following the ICFGF administration. Clinically significant improvement is readilydemonstrable by objective criterion (ETT and/or SAQ) 30 days followingIC administration of the unit dose. In certain patients with progressiveCAD disease, it may be necessary or appropriate to administer a unitdose of the FGF, for example, every six months or annually, to overcomethe progression of the CAD during that interim period.

One of the benefits of the method of the present invention is cardiacangiogenesis. Accordingly, in another aspect, the present invention isdirected to a method for inducing angiogenesis in a heart of a humanpatient, comprising administering as a unit dose into one or morecoronary vessels of a human patient in need of coronary angiogenesisabout 0.2 μg/kg to about 36 μg/kg (or in absolute terms about 0.008 mgto about 6.1 mg) of a recombinant FGF of any one of SEQ ID NOS:1-3, 5,8-10, or 12-14 or an angiogenically active fragment or mutein thereof.

Fifty-two (52) human patients diagnosed with CAD, who satisfied thecriteria of Example 2 herein, were administered a unit dose of 0.33μg/kg to 48 mg/kg of the FGF-2 of SEQ ID NO:5 by IC infusion over abouta 20 minute period. The 52 treated patients were then assessed by theSeattle Angina Questionnaire, which provides an assessment based upon amixed combination of objective and subjective criteria. See Table 2. TheSeattle Angina Questionnaire is a validated, disease-specific instrumentwith the following five subscales that are assessed both before andafter treatment: 1) “exertional capacity”=limitation of physicalactivity; 2) “disease perception”=worry about MI; 3) “treatmentsatisfaction”; 4) “angina frequency”=number of episodes and sublingualnitroglycerin usage; and 5) “angina stability”=number of episodes withmost strenuous physical activity. The possible range for each of thefive subscales is 0 to 100 with the higher scores indicating a betterquality of life. Moreover, a mean change of 8 points or more between themean baseline scores (before treatment) and the post-treatment scores isrecognized as being “clinically significant.” Table 2 reports that the28 patients, who were pretested and then administered a single unit doseof 0.33 μg/kg to 24 μg/kg of the FGF-2 of SEQ ID NO:5 by IC infusion,exhibited a mean score increase of 13 to 36 points for the five “qualityof life” criteria assessed by the “Seattle Angina Questionnaire.” SeeTable 2 herein. These 13 to 36 point increases were about 1.6 to 4.5times greater than the 8 point change which is recognized in the art asbeing “clinically significant” in alternative modes of treatment. SeeTable 2 herein. Moreover, when the combined results for the first 15patients of Table 2 were broken down between low dose (less than orequal to 2 μg/kg) and high (more than 2 μg/kg) doses of FGF-2 of SEQ IDNO:5, and assessed by the “Seattle Angina Questionnaire,” both doseswere found to provide increased scores that ranged from about 12.3 to58.1 and about 10.9 to 32.1, respectively. See Table 3 herein. Theincreased scores were about 1.4 to 7.2 times greater than the 8 pointchange which is considered to be “clinically significant” in alternativemodes of treatment.

In the same Phase I trial, fifty two human patients who were diagnosedwith CAD and who satisfied the criteria of Example 2 herein, wereadministered IC a single unit dose of 0.33 μg/kg to 48 μg/kg of FGF-2 ofSEQ ID NO:5. The maximum tolerated dose (MTD) in humans was defined as36 μg/kg based upon the occurrence of severe but transient hypotensionin 2/10 patients at 48 μg/kg. (In contrast, the MTD in pigs was definedas 6.5 μg/ml.) At one of the sites, the hearts of 23 human patients wereassessed both before (“baseline”) and 30 and 60 days after treatment bymagnetic resonance imaging (MRI) for objective signs of improvedcoronary sufficiency. Among the objective criteria assessed by MRI arethe following: 1) left ventricular (LV) ejection fraction (EF); 2)normal wall thickness (NWT); 3) normal wall motion (NWM); 4) collateralextent; 5) ischemic area zone; 6) targeted wall thickness (TWT); 7)targeted

TABLE 2 COMPARISON OF QUALITY OF LIFE BEFORE AND 57 DAYS AFTER IC FGF-2Seattle Angina Baseline 57 Days Post Questionnaire (SAQ) (Pre FGF-2)FGF-2 Mean Mean p Subscales Mean Score ± SD Score ± SD Change¹ Value nExertional Capacity 55 ± 23 68 ± 25 13* 0.02 28 Angina Frequency 42 ± 3266 ± 28 24* <0.001 28 Angina Stability 46 ± 26 82 ± 20 36* <0.001 27Disease Perception 40 ± 21 61 ± 26 19* <0.001 28 Treatment Satisfaction74 ± 24 88 ± 16 14* 0.002 28 *Significantly different from baseline tofifty-seven days. ¹A mean change of 8 points or more is consideredclinically significant.

TABLE 3 IMPROVEMENTS IN THE QUALITY OF LIFE AT DAY 57 (POST IC rFGF-2)AT LOWER AND HIGHER DOSES Seattle Angina Questionnaire (SAQ) SubscalesDose <2 μg/kg IC Dose >2 μg/kg IC rFGF-2 (n = 7) rFGF-2 (n = 8) MeanChange Mean Change in in Score (Day 57 Score (Day 57 IndependentSubscales score-screen score) score-screen score) Samples t-testExertional Capacity 12.30 (23.3) 15.98 (28.7) t = −.27 p = .79 DiseasePerception 26.19 (26.9) 24.47 (21.2) t = .14 p = .89 Treatment 22.32(27.7) 10.93 (17.3) t = .97 p = .35 Satisfaction Angina Frequency 28.57(27.3) 13.75 (22.6) t = 1.15 p = .27 Angina Stability 58.13 (12.9) 32.14(34.5) t = 1.75 p = .108 1. Possible range for each subscale is 0 to 100with higher scores indicating better quality of life. 2. Standarddeviation noted in parentheses.wall motion (TWM); and 8) perfusion or delayed arrival zone (% LV). Thepatients were also assessed for angina, treadmill exercise duration,rest/exercise nuclear perfusion. The results are summarized in Table 4.Table 4 reflects that the baseline angina class decreased from 2.6 to1.4 and 1.2 at 30 and 60 days, respectively post IC FGF-2. The meantreadmill exercise time increased from a baseline of 8.5 minutes to 9.4and 10.0 minutes at 30 and 60 days, respectively, post treatment. Nosignificant difference was observed in the left ventricular ejectionfraction (LV EF). However, the target wall motion increasedsignificantly, moving from a baseline of 15.4% to 23.5% (day 30) and24.1% (day 60) post FGF-2 treatment. Likewise the target wall thickeningincreased significantly from a baseline of 28.7% to 34.7% (day 30) and45.9% (day 60) post FGF-2 treatment. There was also a significantincrease in perfusion, as measured by a decrease in the delayed arrivalzone (% LV), with the delayed arrival zone decreasing from a baseline of18.9% to 7.1% (day 30) and 1.82% (day 60) post FGF-2 treatment. Thus,providing CAD patients with a single IC infusion of FGF-2 in accordancewith the present invention provided the patients with a significantphysical improvement as objectively measured by MRI and otherconventional criteria.

Pharmacokinetics and Metabolism

The molecular structure of FGF-2 contains a positively charged tail thatis known to bind to proteoglycan chains (heparin and heparin-likestructures) on cell surfaces and on the endothelial wall of thevasculature. See Moscatelli, et al., “Interaction of Basic FibroblastGrowth Factor with Extracellular Matrix and Receptors,” Ann. NY Acad.Sci., 638:177-181 (1981). Because the endothelium is responsible forbinding FGF-2 and acts as a sink after injection, we believed thatrFGF-2 will undergo a fast biodistribution phase right afteradministration. Accordingly, we targeted the intracoronary as opposed tothe intravenous route of administration.

The kidneys and liver are the major organs for the elimination ofrFGF-2. In particular, the kidneys have a protein cutoff of about 60 kDand thus retain serum albumin (MW 60 kD). However, the FGF-2 of SEQ IDNO:5 has a molecular weight of

TABLE 4 MEAN DATA AND DATA RESULTS AS A FUNCTION OF TIME AND DOSEBaseline 30 Day 60 Day Angina Class 2.6 ± 0.7 1.4 ± 0.9***  1.2 ± 0.8***Exercise Time (min.) 8.5 ± 2.6 9.4 ± 1.9*** 10.0 ± 2.5**  LV EF (%) 47.4± 12.3 47.4 ± 10.6   48.6 ± 11.0  Target Wall 15.4 ± 10.1 23.5 ± 12.0* 24.1 ± 10.1** Motion (%) Target Wall 28.7 ± 14.0 34.7 ± 14.1   45.9 ±11.7** Thickening (%) Delayed Arrival 18.9 ± 8.3  7.1 ± 3.6*** 1.82 ±2.4*** Zone (% LV) *= p < 0.05 **= p < 0.01 ***= p < 0.001 (2-tailed,paired)about 16 kD. Accordingly, renal excretion is to be expected. In aradiolabelled biodistribution study of commercially available bovineFGF-2 (bFGF-2), both the liver and the kidney were shown to contain highcounts of the radiolabelled bFGF-2 at 1 hour after IV or IC injection.In the same study, FGF-2 appeared to bind to red blood cells, howeverthese results were not confirmed by in vitro analysis of the wholeblood. In a published study, wherein another recombinant iodinated formof bFGF-2 was given to rats, the liver was identified as the major organof elimination. Whalen et al., “The Fate of Intravenously AdministeredbFGF and the Effect of Heparin,” Growth Factors, 1:157-164 (1989). Moreparticularly, it is known that FGF-2 binds in the general circulation toα₂-macroglobulin and that this complex is internalized by receptors onthe Kupffer cells. Whalen et al. (1989) and LaMarre et al., “CytokineBinding and Clearance Properties of Proteinase-ActivatedAlpha-2-Macroglobulins,” Lab. Invest., 65:3-14 (1991). Labelled FGF-2fragments were not found in the plasma, but they were found in the urineand corresponded in size to intracellular breakdown products. When FGF-2was administered in combination with heparin, the renal excretion ofFGF-2 was increased. Whalen et al. (1989). The FGF-2 molecule, which iscationic when not complexed with heparin, is likely repelled by thecationic heparin sulfate of the glomerular basement membrane. TheFGF-2/heparin complex is more neutrally charged, and therefore is moreeasily filtered and excreted by the kidney.

We determined the pharmacokinetics of rFGF-2 (SEQ ID NO:5) afterintravenous (IV) and intracoronary (IC) administration in domesticYorkshire pigs, after IV dosing in Sprague Dawley (“SD”) rats, and afterIC administration in CAD human patients. In all species, the rFGF-2plasma concentrations after IV and/or IC injection followed abiexponential curve with an initial steep slope and considerabledecrease over several log scales (the distribution phase) during thefirst hour, followed by a more moderate decline (the elimination phase).FIG. 1 provides a plasma concentration versus time curve showing thesephases in humans after IC administration of rFGF-2 of SEQ ID NO:5 as afunction of the following doses: 0.33 μg/kg, 0.65 μg/kg, 2 μg/kg, 6μg/kg, 12 μg/kg, and 24 μg/kg of lean body mass (LBM). The plasmaconcentrations of rFGF-2 of SEQ ID NO:5 were determined by acommercially available ELISA (R &D Systems, Minneapolis, Minn.) that wasmarketed for analysis of human FGF-2. The ELISA assay showed 100%cross-reactivity with the rFGF-2 of SEQ ID NO:5. Other members of theFGF family, as well as many other cytokines, were not detected by thisassay. Further, heparin does not interfere with the assay.

The design of these pharmokinetic studies, pharmacokinetic parameters,and conclusions are listed in Tables 5 and 6 for studies in pigs andrats, respectively. The reader is referred to these tables for thespecific details. However, among the points to be noted are that thehalf-life (T_(1/2)) was 2.8±0.8 to 3.5 hours following a single ICinfusion for the single component model for animals having a clearance(CL) of 702±311 to 609±350 ml/hr/kg. The results of this study show thatthe pharmacokinetics of the recombinant bFGF-2 of SEQ ID NO:5 weresubstantially identical regardless of whether the animals were dosed viathe IC or IV routes. See Table 5. In pigs, the maximum tolerated dose ofrecombinant bFGF-2 was 6.5 μg/kg. Among the other pharmacokineticresults to be taken from Tables 5 and 6 of these studies is that thereis a fast distribution phase followed by a more moderate eliminationphase, and dose linearity as reported in FIG. 1 for humans. Also, therewere no gender differences. Further, the three compartment model wasanalyzed for pigs receiving 70 U/kg of heparin approximately (“˜”) 15minutes before receiving 0.65-6.5 μg/kg by 5-10 minute IC infusion. Thehalf lives (T_(1/2α), T_(1/2β) and T_(1/2γ)) for the three compartmentswere 1.5 minutes, 17 minutes, and 6.6 hours, respectively. In theseanimals, the initial volume (“V₁”) was approximately the plasma volume,and the steady state volume (“V_(ss)”) was approximately 10-fold theplasma volume. See Table 5. In pigs, the binding of recombinant bFGF-2of SEQ ID NO:5 to circulating heparin appears to decreasebiodistribution and elimination. Likewise, in rats, both the volume ofdistribution and the clearance of rFGF-2 were smaller when heparin wasadministered. See Table 6. Further, the greatest and most favorablechanges on clearance of FGF-2 were found when heparin was administeredwithin ±15 minutes, preferably immediately prior to rFGF-2 IC infusion.See Table 6.

TABLE 5 Pharmacokinetics (PK) and Pharmacodynamics of rFGF-2 in PigsAnimals Dosing Regimen PK Parameters Results Domestic Yorkshire pigs2-20 μg/kg IV bolus CL = 702 ± 311 mL/hr/kg Systemic PK identicalbetween IV and IC route under general anesthesia 2-20 μg/kg IC bolus T½= 2.8 ± 0.8 hr. Fast distribution phase (n = 13; 30 ± 5 kg) 20 μg/kg by10-min IC Dose-linearity infusion Transient decreases of MAP 70 U/kgheparin ~15 min before rFGF-2 Domestic Yorkshire pigs 0.65-6.5 μg/kg by5-min CL = 609 ± 350 ml/hr/kg No gender difference in PK under generalanesthesia IC infusion T½ = ~3.5 hr Biphasic decline of plasma rFGF-2 (n= 17; 26 ± 4 kg) 70 U/kg heparin ~15 3-Comp. Model: Dose-linearity minbefore rFGF-2 T½α = 1.5 min V₁ equal to ~ plasma volume T½β = 17 minV_(ss) equal to ~ 10-fold plasma volume T½γ = 6.6 hr Magnitude andduration of MAP decrease correlated CL = 580 ml/hr/kg with rFGF-2 doseand peak plasma level V₁ = 55 ml/kg V_(ss) = 523 ml/kg DomesticYorkshire pigs 6.5 μg/kg weekly by 5 Without Heparin The rFGF-2distribution phase was less steep, the under general anesthesia min IVinfusion for 6 (Doses 1-6): volume of distribution smaller, andclearance was (n = 6; 25 ± 5 kg) weeks T½ = 2-6 hr slower withheparin-pretreatment 70 U/kg heparin 10 min CL = 777-2749 ml/hr/kgBinding of rFGF-2 to circulating heparin appears to before rFGF-2 (n =3), or V_(ss) = 871-12,500 ml/kg decrease biodistribution andelimination rFGF-2 alone (n = 3) With Heparin Both volume and clearanceof rFGF-2 increased at (Doses 1-6): later doses (potential receptorupregulation), but more T½ = 2-3 hr so in the absence of heparin CL =235-347 ml/hr/kg Magnitude and duration of MAP decreases were V_(ss) =71-153 ml/kg similar with or without heparin

TABLE 6 Pharmncokinctics (PK) of rFGF-2 in Rats Animals Dosing RegimenPK Parameters Results Conscious SD rats 3-100 μg/kg bolus IV T½ = 1.1 ±0.51 hr Fast distribution phase (n = 18; 322 ± 93 g) injection CL = 4480± 2700 ml/hr/kg Apparent dose-linearity V_(ss) = 1924 ± 1254 ml/kgconscious SD rats 30-300 μg/kg weekly by T½ = 1.4 ± 0.13 hrTime-invariant PK; plasma profiles, PK parameters and (n = 54; 149 ± 12g) bolus IV injection for 6 CL = 1691 ± 169 ml/hr/kg AUCs were similarover time weeks V_(ss) = 1942 ± 358 ml/kg Dose-linearity No heparinpretreatment Conscious SD rats 30 μg/kg bolus IV Time-Averaged PK In allcases, heparin increased the rFGF-2 plasma levels (27 males; 381 ± 48 g;injection Parameters: Both volume of distribution and clearance of 20females; 268 ± 22 g) No heparin T½ CL V_(ss) rFGF-2 were smaller withheparin 40 U/kg IV Heparin: hr. ml/hr/kg ml/kg Greatest changes on CLand V_(ss) were observed when at ~15 min 0.75 4332 2389 heparin wasadministered immediately prior to rFGF-2 just prior to rFGF-2 0.91 1728844 at +15 min 1.3 516 147 at +3 hr 1.2 1158 626 0.93 1338 1351

The pharmacokinetics of the rFGF-2 of SEQ ID NO:5 was studied in humans,diagnosed with CAD despite optimal medical management, in a Phase 1clinical study supporting this filing. The doses of rFGF-2 employed inthat Phase 1 study were 0.33 μg/kg, 0.65 mg/kg, 2 μg/kg, 6 μg/kg, 12mg/kg, and 24 μg/kg of lean body mass (LBM), and all doses wereadministered by a 20 minute IC infusion (10 minutes into each of twopatent coronary vessels) after pretreating the patient with 40 U/kgheparin which was administered IV or IC 1-95 minutes before rFGF-2infusion. FIGS. 1-3 herein summarize the data underlying those results.In particular, FIG. 1 is a plot of the mean rFGF-2 plasma concentrationversus time (hours) for the six different doses of rFGF-2 (SEQ ID NO:5)administered by IC infusion as described above over a 20 minute period.FIG. 1 shows dose linearity and a biphasic plasma level decline, i.e., afast distribution phase during the first hour, followed by anelimination phase with T_(1/2) of 1.9±2.2 hours. The dose linearity ismore readily seen in FIG. 2, which is a plot of the individual patientrFGF-2 area under the curve (AUC) in pg·hr/ml for FIG. 1 for each of thesix doses of rFGF-2 administered. FIG. 3 is a plot individual humanpatient rFGF-2 dose normalized AUCs versus time of heparin dose in“minutes prior to rFGF-2 infusion” and shows the influence of timing ofheparin administration on rFGF-2 AUC. FIG. 3 shows that the greatestAUC/dose was achieved when an effective amount of a glycosoaminoglycan,such as heparin, was preadministered within 30 minutes or less of ICrFGF-2 infusion, more preferably within 20 minutes or less of IC rFGF-2infusion. Typically, an effective amount of a glycosoaminoglycan is40-70 U/kg heparin. These pharmacokinetic results are summarized inTable 7 herein.

The rFGF-2 distribution phase was less steep with heparin, the volume ofdistribution smaller, and the clearance slower, as compared to rFGF-2without heparin. It appears that the complex of rFGF-2 with circulatingheparin decreases the biodistribution and elimination of rFGF-2.Although the binding of FGF-2 to heparin-like structures is strong(dissociation constant ˜2×10⁻⁹ M), the binding of FGF-2 to the FGF-2receptor is approximately two orders of magnitude higher (dissociationconstant ˜2×10⁻¹¹ M). Moscatelli et al., (1991).

TABLE 7 Pharmacokinetics of rFGF-2 in Human Subjects Dosing Regimen PKParameters Results Patients with CAD 0.33-24 μg/kg LBM* Preliminary PKdata Biphasic plasma level decline: fast distribution by 20-min ICinjection from 0.33-24 μg/kg phase during 1 hr; followed by eliminationphase with (10 min in left main doses (n = 32) T½ approximately (~) 2 hrcoronary artery + 10 T½ = 1.9 ± 2.2 hr Dose-linearity min in right mainCL = 264 ± 150 ml/hr/kg Greater rFGF-2 exposure (as measured by thecoronary artery) V_(ss) = 184 ± 74 ml/kg AUC) was found when heparinpreteatment was 0.33 μg/kg, n = 4 given closer to the start of therFGF-2 infusion, 0.65 μg/kg, n = 4 preferably within 20 minutes 2 μg/kg,n = 8 6 μg/kg, n = 4 12 μg/kg, n = 4 24 μg/kg, n = 8 36 μg/kg, n = 10 48μg/kg, n = 10 40 U/kg heparin pretreatment, 1-95 min before rFGF-2infusion *LBM = lean body mass

In addition, the complexation of the rFGF-2 of SEQ ID NO:5 with aglycosoaminoglycan, such as a heparin, might increase signaltransduction and mitogenesis, and/or protect the rFGF-2 from enzymaticdegradation.

The examples, which follow, provide more details on the selectioncriterion and the Phase I clinical trial that gave rise to the datadiscussed above.

Example 1 Unit Dose of rFGF-2 Employed in the Phase I Clinical Trial

The rFGF-2 of SEQ ID NO:5 was formulated as a unit dose andpharmaceutical composition and administered to rats, pigs, andultimately to humans in the Phase I clinical trial referenced herein.The various formulations are described below.

The rFGF-2 Unit Dose was provided as a liquid in 3 cc type I glass vialswith a laminated gray butyl rubber stopper and red flip-off overseal.The rFGF-2 unit dose contained 1.2 ml of 0.3 mg/ml rFGF-2 of SEQ ID NO:5in 10 mM sodium citrate, 10 mM monothioglycerol, 1 mM disodium dihydrateEDTA (molecular weight 372.2), 135 mM sodium chloride, pH 5.0. Thus, inabsolute terms, each vial (and unit dose) contained 0.36 mg rFGF-2. Thevials containing the unit dose in liquid form were stored at 2° to 8° C.The rFGF Diluent was supplied in 5 cc type I glass vials with alaminated gray butyl rubber stopper and red flip-off overseal. TherFGF-2 diluent contains 10 mM sodium citrate, 10 mM monothioglycerol,135 mM sodium chloride, pH 5.0. Each vial contained 5.2 ml of rFGF-2diluent solution that was stored at 2° to 8° C.The rFGF-2 Pharmaceutical Composition that was infused was prepared bydiluting the rFGF-2 unit dose with the rFGF diluent such that theinfusion volume is 10 ml. In order to keep the EDTA concentration belowthe limit of 100 μg/ml, the total infusion volume was increased to 20 mlwhen proportionately higher absolute amounts of FGF-2 were administeredto patients with high body weights.

Example 2 Selection Criteria for Patients with Coronary Artery Diseasefor Treatment with rFGF-2

The following selection criteria were applied to Phase I patients withcoronary artery disease, whose activities were limited by coronaryischemia despite optimal medical management, and who were not candidatesfor approved revascularization therapies:

Inclusion Criteria:

Subject is eligible if:

-   -   Male or female, greater than or equal to 18 years of age    -   Diagnosis of coronary artery disease (CAD)    -   Suboptimal candidates for approved revascularization procedures,        e.g., angioplasty, stents, coronary artery bypass graft (CABG)        (or refuses those interventions)    -   Able to exercise at least three minutes using a modified Bruce        protocol and limited by coronary ischemia    -   Inducible and reversible defect of at least 20% myocardium on        pharmacologically stressed thallium sestamibi scan    -   CBC, platelets, serum chemistry within clinically acceptable        range for required cardiac catheterization    -   Normal INR, or if anticoagulated with Coumadin, INR<2.0    -   Willing and able to give written informed consent to participate        in this study, including all required study procedures and        follow-up visits

Exclusion Criteria:

Subject is not eligible if:

-   -   Malignancy: any history of malignancy within past ten years,        with the exception of curatively treated basal cell carcinoma    -   Ocular conditions: proliferative retinopathy, severe        non-proliferative retinopathy, retinal vein occlusion, Eales'        disease, or macular edema or funduscopy by ophthalmologist:        history of intraocular surgery within six months    -   Renal function: creatinine clearance below normal range adjusted        for age; protein>250 mg or microalbumin>30 mg/24 h urine    -   Class IV heart failure (New York Heart Association)    -   Ejection fraction<20% by echocardiogram, thallium scan, MRI or        gated pooled blood scan (MUGA)    -   Hemodynamically relevant arrhythmias (e.g., ventricular        fibrillation, sustained ventricular tachycardia)    -   Severe valvular stenosis (aortic area<1.0 cm², mitral area<1.2        cm²), or severe valvular insufficiency    -   Marked increase in angina or unstable angina within three weeks    -   History of myocardial infarction (MI) within three months    -   History of transient ischemic attack (TIA) or stroke within six        months    -   History of CABG, angioplasty or stent within six months    -   History of treatment with transmyocardial laser        revascularization, rFGF-2, or vascular enodothelial growth        factor (VEGF) within six months    -   Females of child-bearing potential or nursing mothers    -   Any pathological fibrosis, e.g., pulmonary fibrosis, scleroderma    -   Known vascular malformation, e.g., AV malformation, hemangiomas    -   Coexistence of any disease which might interfere with assessment        of symptoms of CAD, e.g., pericarditis, costochondritis,        esophagitis, systemic vasculitis, sickle cell disease    -   Coexistence of any disease which limits performance of modified        Bruce protocol exercise stress test, e.g., paralysis or        amputation of a lower extremity, severe arthritis or lower        extremities, severe chronic obstructive pulmonary disease (COPD)    -   Participation in clinical trials of investigational agents,        devices or procedures within thirty days (or scheduled within        sixty days of study drug)    -   Known hypersensitivity to rFGF-2 or related compounds    -   Any condition which makes the subject unsuitable for        participation in this study in the opinion of the investigator,        e.g., psychosis, severe mental retardation, inability to        communicate with study personnel, drug or alcohol abuse

Example 3 Phase I Clinical Study on Recombinant FGF-2 (SEQ ID NO:1)Administered to Humans

Recombinant bFGF-2 of SEQ ID NO:5 was administered to 52 human patientswith severe CAD, who remained symptomatic despite optimal medicalmanagement and who refused or were suboptimal candidates for surgical orpercutaneous revascularization, in a Phase I open label, singleadministration, dose escalation, two-site trial. The drug wasadministered as a single 20 minute infusion divided between two majorsources of coronary blood supply (IC), using standard techniques forpositioning a catheter into the patient's coronary artery (such asalready employed in angioplasty). The doses (μg/kg) of rFGF-2administered were 0.33 (n=4), 0.65 (n=4), 2.0 (n=8), 6.0 (n=4), 12.0(n=4), 24 (n=8), 36 (n=10), and 48 (n=10) of rFGF-2 of SEQ ID NO:5.Angina frequency and quality of life was assessed by the Seattle AnginaQuestionnaire (SAQ) at a baseline (before rFGF-2 administration) and atabout 60 days after rbFGF-2 administration. Exercise tolerance time(ETT) was assessed by the threadmill test. Rest/exercise nuclearperfusion and gated sestamibi-determined rest ejection fraction (EF),and magnetic resonance imaging (MRI) were assessed at baseline, and at30 days and 60 days post FGF-2 administration. Other end points thatwere evaluated included MRI (to objectively measure ejection fraction(EF), normal wall motion (NWM), targeted wall motion (TWM), normal wallthickness (NWT), targeted wall thickness (TWT), ischemic area zone andcollateral extent). See Tables 2-4, respectively.

The preliminary safety results indicate that serious events were notdose related. Thus far, of the eight dosage groups, there were threedeaths in the lowest dosage groups, i.e., at 0.65 μg/kg (Day 23), at 2.0μg/kg (Day 57), and at 6.0 μg/kg (Day 63). There were sixhospitalizations for acute myocardial infarction (MI) in three patients,i.e., one patient from each of groups 1 (0.33 μg/kg), 3 (2.0 μg/kg), and4 (6.0 μg/kg). One of the three patients accounted for four of the sixhospitalizations for acute MI. There was also one large B cell lymphomathat was diagnosed three weeks after dosing in a patient in group 4. Thepatient died at two months post dosing. Acute hypotension, seen athigher doses during or just subsequent to infusion, was managed byadministration of fluids without need for a vasopressor. The maximumtolerated dose of rFGF-2 (SEQ ID NO:2) was defined as 36 μg/kg. Doses ofrFGF-2 up to 48 μg/kg IC were managed in patients with aggressive fluidmanagement. However, they were not tolerated due to acute and/ororthostatic hypotension in two out of ten patients. The half-life inhumans of the IC infused rFGF-2 was about one hour.

The human patients in this study that were treated with a single ICinfusion of rFGF-2 of SEQ ID NO:5 exhibited a mean increase in ETT of1.5 to 2 minutes. This is especially significant because an increase inETT of >30 seconds is considered significant and a benchmark forevaluating alternative therapies, such as angioplasty. The anginafrequency and quality of life, as measured by SAQ, showed a significantimprovement at 57 days in all five subscales for the 28 patients (n=28)tested. See Tables 2 and 3. In particular, the mean changes in scoresfor the five criteria evaluated by the SAQ ranged from 13 to 36 with amean change of 8 or more considered “clinically significant.” See Table2.

Magnetic resonance imaging (MRI) showed objective improvements followingadministration of a single unit dose of the bFGF-2 of SEQ ID NO:5,including increased targeted wall motion at 30 and 60 days (p<0.05), andincreased targeted wall thickening at 60 days (p<0.01). MRI furthershowed improved regional wall motion, and increased myocardial perfusionand collateral development in the targeted area for both the lower dose(0.33 μg/kg and 0.65 μg/kg) and higher dose (2.0 μg/kg and 12.0 μg/kg)groups in an 11 patient test group (n=11).

Abnormal perfusion zone, which was assessed at one of the sites on 28patients, decreased significantly at 30 and 60 days (p<0.001).

In addition to the above criterion (i.e., ETT SAQ, MRI), a treatment isconsidered very successful if the angiogenic effects last at least sixmonths. In the present Phase I study, the unexpectedly superiorangiogenic effects were observed to last for 57-60 days in all dosagegroups. (See Tables 2-4.) Based upon the results already obtained, it isexpected that the angiogenic effects would last twelve months or morebut at least six months, at which time the procedure could be repeated,if necessary.

Example 4 Proposed Phase II Clinical Study on Recombinant FGF-2 (SEQ IDNO:1) Administered to Humans to Treat Coronary Artery Disease

The Phase II clinical trial of rFGF-2 for treating human patients forcoronary artery disease is performed as a double blind/placebocontrolled study having four arms: placebo, 0.3 μg/kg, 3 μg/kg, and 30μg/kg administered IC.

Example 5 Unit Dose and Pharmaceutical Composition of rFGF-2 for thePhase II Human Clinical Trial

The rFGF-2 of SEQ ID NO:5 was formulated as a unit dose andpharmaceutical composition for administration to humans in the Phase IIclinical trial referenced herein. The various formulations are describedbelow.

The rFGF-2 Unit Dose was prepared as a liquid in 5 cc type I glass vialswith a laminated gray butyl rubber stopper and red flip-off overseal.The rFGF-2 formulation contains 0.3 mg/ml rFGF-2 of SEQ ID NO:5 in 10 mMsodium citrate, 10 mM monothioglycerol, 0.3 mM disodium dihydrate EDTA(molecular weight 372.2), 135 mM sodium chloride, pH 5.0. Each vialcontained 3.7 ml of rFGF-2 drug product solution (1.11 mg rFGF-2 pervial). The resulting unit dose in liquid form is stored at less than−60° C. The above described unit dose is diluted with the “rFGF-2placebo.” Depending on the size of the patient, the contents of severalof the vials may be confirmed to produce a unit dose of 36 mg/kg for thePhase II study.The rFGF Placebo is supplied as a clear colorless liquid in 5 cc type Iglass vials with a laminated gray butyl rubber stopper and red flip-offoverseal. The rFGF-2 placebo is indistinguishable in appearance from thedrug product and has the following formulation: 10 mM sodium citrate, 10mM monothioglycerol, 0.3 mM disodium dihydrate EDTA (molecular weight372.2), 135 mM sodium chloride, pH 5.0. Each vial contains 5.2 ml ofrFGF-2 placebo solution. Like the unit dose, the rFGF-2 placebo isstored at 2° to 8° C.The rFGF-2 Pharmaceutical Composition that is infused is prepared bydiluting the rFGF-2 unit dose with the rFGF diluent such that theinfusion volume is 20 ml for Phase II.

Example 6 Selection Criteria for CAD Patients for the Phase II HumanClinical Trial of IC rFGF-2

Accordingly, the above described evidence of an unexpectedly superiorimprovement in quality of life and of increased angiogenic efficacy inhumans who were administered a single unit dosage of rFGF-2 inaccordance with this invention, supports the patentability of theApplicants' unit dose, pharmaceutical composition and method of usingthe same.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference in its entirety.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the present invention described herein.

That which is claimed is:
 1. A method for treating a human patient forcoronary artery disease, comprising administering into one or morecoronary vessels in a human patient in need of treatment for coronaryartery disease a therapeutically effective amount of an angiogenicallyactive fragment or angiogenically active mutein of a recombinantfibroblast growth factor (FGF) having the sequence set forth in SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:9.
 2. The method of claim1, wherein said therapeutically effective amount administered to saidpatient is a unit dose of about 0.008 mg to about 6.1 mg of saidangiogenically active fragment or angiogenically active mutein of saidrecombinant FGF.
 3. The method of claim 2, wherein said therapeuticallyeffective amount administered to said patient is a unit dose of 0.3 mgto 3.5 mg of said angiogenically active fragment or angiogenicallyactive mutein of said recombinant FGF.
 4. The method of claim 1,comprising administering into one or more coronary vessels of saidpatient about 0.2 μg/kg to about 36 μg/kg of said angiogenically activefragment or angiogenically active mutein of said recombinant FGF.
 5. Themethod of claim 4, comprising administering into one or more coronaryvessels of said patient about 0.2 μg/kg to about 2 μg/kg of saidangiogenically active fragment or angiogenically active mutein of saidrecombinant FGF.
 6. The method of claim 4, comprising administering intoone or more coronary vessels of said patient about 2 μg/kg to about 20μg/kg of said angiogenically active fragment or angiogenically activemutein of said recombinant FGF.
 7. The method of claim 4, comprisingadministering into one or more coronary vessels of said patient about 20μg/kg to about 36 μg/kg of said angiogenically active fragment orangiogenically active mutein of said recombinant FGF.
 8. A method fortreating a human patient for coronary artery disease, comprisingadministering into one or more coronary vessels in a human patient inneed of treatment for coronary artery disease a therapeuticallyeffective amount of a recombinant fibroblast growth factor (FGF) havingthe sequence set forth in SEQ ID NO:10 or 12-14 or an angiogenicallyactive fragment or an angiogenically active mutein thereof.
 9. Themethod of claim 8, wherein said therapeutically effective amountadministered to said patient is a unit dose of about 0.008 mg to about6.1 mg of said recombinant FGF or said angiogenically active fragment orsaid angiogenically active mutein thereof.
 10. The method of claim 9,wherein said therapeutically effective amount administered to saidpatient is a unit dose of 0.3 mg to 3.5 mg of said recombinant FGF orsaid angiogenically active fragment or said angiogenically active muteinthereof.
 11. The method of claim 8, comprising administering into one ormore coronary vessels of said patient about 0.2 μg/kg to about 36 μg/kgof said recombinant FGF or said angiogenically active fragment or saidangiogenically active mutein thereof.
 12. The method of claim 11,comprising administering into one or more coronary vessels of saidpatient about 0.2 μg/kg to about 2 μg/kg of said recombinant FGF or saidangiogenically active fragment or said angiogenically active muteinthereof.
 13. The method of claim 11, comprising administering into oneor more coronary vessels of said patient about 2 μg/kg to about 20 μg/kgof said recombinant FGF or said angiogenically active fragment or saidangiogenically active mutein thereof.
 14. The method of claim 11,comprising administering into one or more coronary vessels of saidpatient about 20 μg/kg to about 36 μg/kg of said recombinant FGF or saidangiogenically active fragment or said angiogenically active muteinthereof.
 15. A method for inducing angiogenesis in the heart of apatient, comprising administering into one or more coronary vessels of ahuman patient in need of coronary angiogenesis a therapeuticallyeffective amount of a recombinant fibroblast growth factor (FGF) havingthe sequence set forth in SEQ ID NO:3, 5, 10 or 12-14 or anangiogenically active fragment or an angiogenically active muteinthereof, or a therapeutically effective amount of an angiogenicallyactive fragment or an angiogenically active mutein of a recombinantfibroblast growth factor (FGF) having the sequence set forth in SEQ IDNO:8 or SEQ ID NO:9.
 16. The method of claim 15, wherein saidtherapeutically effective amount administered to said patient is a unitdose of about 0.008 mg to about 6.1 mg of said recombinant FGF or saidangiogenically active fragment or said angiogenically active muteinthereof.
 17. The method of claim 15, wherein said unit dose isadministered into two coronary vessels of said patient.
 18. The methodof claim 15, further comprising the step of administering to saidpatient an effective amount of a glycosaminoglycan within 30 minutes ofadministering said recombinant FGF or said angiogenically activefragment or said angiogenically active mutein thereof.
 19. A unit doseof a fibroblast growth factor (FGF), comprising about 0.008 mg to about6.1 mg of an FGF having the amino acid sequence of SEQ ID NO:1-3, 5,8-10 or 12-14 or an angiogenically active fragment or an angiogenicallyactive mutein thereof.
 20. A pharmaceutical composition comprising theunit dose of said FGF according to claim 19, and a pharmaceuticallyacceptable carrier, said composition being in a form and a size suitablefor administration to a human patient.
 21. A pharmaceutical compositioncomprising (i) a therapeutically effective amount of a fibroblast growthfactor (FGF) having the sequence of SEQ ID NO:1-3, 5, 8-10 or 12-14 oran angiogenically active fragment or an angiogenically active muteinthereof, and (ii) a pharmaceutically acceptable carrier, saidcomposition being in a form and a size suitable for administration to ahuman patient, wherein said therapeutically effective amount comprisesabout 0.2 μg/kg to about 36 μg/kg of said FGF or said angiogenicallyactive fragment or said angiogenically active mutein thereof.